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Turk J Pharm Sci 10(1), 1-8, 2013

Original article

QUALITATIVE AND QUANTITATIVE ANALYSIS OF PHENOLIC ACIDS IN SCORZONERA TOMENTOSA L.

ÖzlemBAHADIR ACIKARA1,*, Giilcin SALTAN CITOGLU1,

Ayse Mine GENCLER OZKAN2

1Ankara University, Faculty of Pharmacy, Department of Pharmacognosy, 06100 Ankara, TURKEY 2Ankara University, Faculty of Pharmacy, Department of Botany, 06100 Ankara, TURKEY

Abstract In the present study the qualitative and quantitative analysis of phenolic acids such as chlorogenic CA), caffeic (CFA), ferulic (FA), rosmarinic (RA) and p-coumaric acids (PCA), were performed by using high pressure liquid chromatography in the aerial part and the root extracts of Scorzonera tomentosa L. (Asteraceae) which were collected from five different regions of Anatolia. was determined as major phenolic acid content of Scorzonera tomentosa. The calibration curve was established as Y=23.21X–21.83 (r2= 0.999) for chlorogenic acid and the highest concentration was determined in the root extract of Yozgat sample with 1360.090 ± 6.732 fig.

Key words: Scorzonera tomentosa, Phenolic acid, Chlorogenic acid, HPLC

Scorzonera tomentosa L. Bitkisinde Fenolik Asitlerin Kalitatif ve Kantitatif Analizi

Bu cahsmada Anadolu'nun besfarkh bolgesinden toplanan Scorzonera tomentosa L. (Asteraceae) bitkisinin toprak üstü hsimlari ve köklerinin klorojenik, kafeik, ferulik, rosmarinik ve p-kumarik asit gibi fenolik asit igerikleri kalitatif ve kantitatif olarak yuksek basingh sivi kromatografisi kullamlarak karsilastinlmistir. Tespit edilen major fenolik asit klorojenik asittir. Klorojenik asit için kalibrasyon denklemi Y=23.21X-21.83 (r2= 0.999) olarak bulunmus ve en yuksek klorojenik asit igerigine 1360.090 ± 6.732 fig He Yozgat'tan toplanan ornegin kok ekstresinin sahip oldugu belirlenmistir

Anahtar kelimeler: Scorzonera tomentosa, Fenolik asit, Klorojenik asit, YBSK

Correspondence: E-mail: [email protected]; Tel.: +903122033103

1 Ozlem BAHADIR ACIKARA, Gulgin SALTAN 0TIOGLU, Ay§eMine GENCLER OZKAN

INTRODUCTION

Phenolic acids are one of the important groups of phenolic compounds that are widely distributed in both edible and non edible plants (1-4). They are hydroxylated derivatives of benzoic and sinnamic acids and the most commons are derivatives such as chlorogenic, caffeic, /?-coumaric, ferulic acids (1-2, 5-6). Phenolic acids have attracted considerable interest in the past few years due to their strong antioxidant activities against free radicals and other reactive oxygen species (ROS) which are the cause of many chronic diseases such as cancer, cardiovascular diseases, inflammation, brain dysfunction (4, 7). There are also many literatures related to the different pharmacological activities of phenolic acids including anticancer, antibacterial, antipyretic, antirheumatic activities (2, 4, 7-8). The genus Scorzonera L. (Asteraceae) is widely distributed in the arid regions of Asia, Europe and in Northern Africa with more than 175 species (9-10). In Turkey Scorzonera genus contains 49 species with the addition of new species (10-13). Some species of Scorzonera have been widely used as a food as well as medicinal plants (14-16).Some species of this genus are known as yemlik in Anatolia and their roots as well as green buds are eaten freshly or after cooked. They have also some medicinal usage in Turkish folk medicine for the treatment of arteriosclerosis, kidney diseases, hypertension, diabetes mellitus and rheumatism as well as for pain treatment and wound healing (17-18). Scorzonera tomentosa L. is an endemic herb to Turkey. S. tomentosa roots contain latex, both of this latex and roots are used due to their wound healing activity in Turkish folk medicine (19). Previous studies on Scorzonera genus revealed that many compounds such as dihydroisocoumarines (20-21), bibenzyl derivatives (22-24), (15, 25-27), (24, 28-29), stilbene derivatives (16, 21, 30), quinic and derivatives (15, 27, 30) sesquiterpene, sesquiterpene lactones (15, 22, 28, 31) and triterpenes (16, 27, 32-33) have been isolated. In addition, stigmasterol 3(3-glucoside, (3-sitosterol, lupeol, lupeol acetate and a- amyrine from the aerial parts (32) and hydrangenol, hydrangenol-4'-O-glucoside, hydramacrophyllol A and B as well as scorzotomentosin, scorzotomentosin-4'-O-(3-glucoside, scorzophtalide, scorzoerzincanin from the roots of S. tomentosa (21) hydrangenol and scorzotomentosin 4'-O-(3-glucopyranoside from the roots of S. latifolia (34) have been isolated previously. In our previous studies we have been reported the analgesic activities of S. latifolia. Both plant root extracts and the isolated compounds; taraxasteryl myristate and taraxasteryl acetate showed promising antinociceptive activities (33, 35). This paper is a part of our on going studies on this genus (33-36). The aim of the current study was to give a general profile of the content of phenolic acid variation in S. tomentosa samples which were collected from different regions of Anatolia. Chlorogenic, caffeic, ferulic, /?-coumaric and rosmarinic acid contents of S. tomentosa samples were investigated qualitatively and quantitatively by using RP-HPLC method.

EXPERIMENTAL

Plant material Five Scorzonera tomentosa L. (Asteraceae) species were collected in different locations of Turkey. The species are listed in Table 1. Voucher specimens were deposited at the Herbarium of Ankara University, Faculty of Pharmacy (AEF) and botanical authentication was confirmed by H. Duman a plant taxonomist in the Department of Biological Sciences, Faculty of Art and Sciences, Gazi University and A. Mine Gençler Özkan, Department of Pharmaceutical Botany, Faculty of Pharmacy, Ankara University, Ankara, Turkey.

2 Turk J Pharm Sci 10(1), 1-8, 2013

Chemicals Chlorogenic acid (CA) (C3878-1G), caffeic acid (CFA) (095K1340) and p-coumaric acid (PCA) (C900856) were obtained from Sigma; (FA) (128708) and rosmarinic acid (RA) (536954) were purchased from Aldrich.

General experimental procedures Chromatography was performed on Agilent LC 1100 model chromatograph (Agilent Technologies, California, USA). The diode array detector (DAD) was set at wavelength 330 nm and peak areas were integrated automatically by computer using Agilent Software. The chromatograms were plotted and processed by using the above mentioned software. The column was Supelcosil (25 cm x 4 mm x 5 um) (Hichrom). The column temperature was 25 °C and the mobile phase included water (containing 0.2% phosphoric acid, solvent A) and acetonitrile (solvent B) in the following gradient system: initial 6% B, linear gradient to 30% B in 20 min, hold at 30% for 5 min. The total running time was 25 min. The flow rate was 1.2 mL/min injection volume was 10 uL (37).

Preparation of HPLC samples and standard solutions HPLC samples were prepared from 0.25 g of plant material using stirrer at 50 °C for 60 min using 10 mL of methanol: water (80:20) mixture. After extraction, all samples were adjusted to 10 mL in volumetric flask and filtrated from 0.45 uM filters before HPLC analysis. All standard compounds were dissolved in ACN and water mixture for qualitative analysis. For quantitative analysis, CA (10 mg) was accurately weighed in 10 mL volumetric flask and dissolved in ACN and water mixture then diluted to the final volume separately. Seven different concentrations (0.0025 mg/mL, 0.005 mg/mL, 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL 0.2 mg/mL, 0.3 mg/mL) were prepared by diluting the stock solutions. Triplicate 10 uL injections were performed for both standard solution and plant samples. Peak area of each solution was plotted against the concentration to obtain the calibration curve.

Table 1. Locality of S. tomentosa samples

Plant sample Locality AEF No Collection Date 1 Yozgat-Akdagmadeni 23841 2005 2 Kayseri-Pinarbasi 23838 2005 3 Sivas-Hafik 23840 2005 4 Erzincan 23839 2005 5 Erzurum-Kop Geçidi 25158 2005

Limits of detection and quantification Limits of detection (LOD) were established at a signal to noise ratio (S/N) of 3. Limits of quantification (LOQ) were established at a signal to noise ratio (S/N) of 10. LOQ were experimentally verified by 6 injections of CA at the LOD and LOQ concentrations. LOD and LOQ concentrations were calculated as 0.47 μg/mL, 1.56 μg/mL for CA respectively. The precision of the method was performed by the evaluation of intra-day and inter-day variations of the same standard solution at the LOQ level. The intra-day precision was determined by analyzing the same samples six times in a single day and inter-day precision was determined three different days in triplicates of injections.

3 Ozlem BAHADJR ACJKARA, Giilgin SALTAN 0TJOGLU, Ay§eMine GENTLER OZKAN

The precision of the method expressed as the RSD % at the LOQ level was for CA (Table 2).

Table 2. Precision of the method at the LOQ Level Precision % RSD LOD LOQ Intra-day Inter-day Standards (ug/mL) (ug/mL) (n=6) (n=3) Rt Pa Rt Pa CA 0.47 1.56 0.021 1.133 0.054 1.012

RESULTS AND DISCUSSION

The purpose of the current study was to investigate the content of phenolic acids including CA, CFA, PCA, FA and RA (Fig 1) as well as to obtain a comparison of phenolic acid content amongst S. tomentosa samples from different geographical origins of Anatolia. HPLC results have revealed that CA was determined as the most abundant phenolic acid in both roots (Fig 2) and aerial parts (Fig 3) of S. tomentosa. HPLC chromatographic profiles of all S. tomentosa root and aerial part samples were found to be qualitatively similar among them. However there were significant differences in the level of CA content. S. tomentosa roots contain CA in higher percentage than aerial parts except Kayseri sample and the highest content of CA was determined in the roots of Yozgat-Akdagmadeni sample with 1360.090 ± 6.732 ug/mg as shown in Table 3.

Table 3. Chlorogenic acid contents of S. tomentosa samples Plant Aerial parts (ug/mg) Roots (ug/mg) sample (n=3, mean ±SD) (n=3, mean ±SD) 1 441.497 ± 3.475 1360.090 ± 6.732 2 764.203 ± 0.657 336.557 ± 1.195 3 191.300 ± 1.573 1013.994 ± 3.243 4 293.793 ± 0.949 933.357 ±0.791 5 400.297 ± 1.994 1048.403 ± 4.398

DAD1 C, Sig=330,8 Ref=off (SCORZO\SLST0002.D) mAU - 1 5 2000-

1750- 2 3 4

1500-

1250-

1000-

750-

500-

250- I Ii J \ 0- 1 L I D 5 10 15 20 min

Figure 1. Chromatogram of the standart mixture, 1: Chlorogenic acid; 2: Caffeic acid; 3: p- Coumaric acid; 4: Ferulic acid; 5: Rosmarinic acid

4 Turk J Pharm Sci 10(1), 1-8, 2013

DAD1 C, Slg=330,8 Ref=off (SCORZO\SLST0023.D) mAU

3oO 1

300

ZoU

200

1 oO

I 00

JO

X l.t „ 1 . IJU,A,

u 10 lo :?0 mi

Figure 2. Chromatogram of the root extract of Akdagmadeni sample

DAD1 C, Slg=330,8 Ref=ofT (SCORZO\SLST0026.D) mAU

120

I 00

30

30

■.'

20

0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 mi

Figure 3. Chromatogram of the aerial part extract of Akdagmadeni sample

It has been reported that, several factors including genetic differences, climatic conditions, agricultural practices as well as geographical locations can affect the chemical composition of the plants (38). The results obtained in present study have also confirmed the variation in the phenolic acid content amongst samples from different geographical origin. Different growing conditions have probably influences on the content of CA. CA that is one of the most abundant in human diet is widely found in plant kingdom including fruits and vegetables. CA is known to have strong antioxidant activity. In addition, previous studies have revealed that CA has also many biological activities including anti-inflammatory (39-40), antinociceptive (39, 41) and cut effect on the glucose absorption (42). CA and derivatives have also been identified and isolated from some Scorzonera species (30, 36). Phenolic acids including CA, CFA, CPA, FA, RA and flavonoids such as , luteolin, , luteolin-7-glucoside, rutin, hyperoside and were investigated in Scorzonera species in our earlier study and CA was determined as one of the major constituent of Scorzonera species (36). It has been suggested that CA probably responsible for the biological activities of Scorzonera species in part.

5 Ozlem BAHADIR ACIKARA, Gülgin SALTAN 0TIOGLU, Ay§eMine GENCLER OZKAN

In conclusion present study showed exhibited that Scorzonera tomentosa contains CA as one of the main component in significant amounts. Additionally amount of the CA was found to vary depending on the geographical locations of the plant samples.

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Received: 09.09.2011 Accepted: 24.02.2012

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